Christian Speck

A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication

During pre-replication complex (pre-RC) formation, origin recognition complex (ORC), Cdc6, and Cdt1 cooperatively load the 6-subunit mini chromosome maintenance (MCM2-7) complex onto DNA. Loading of MCM2-7 is a prerequisite for DNA licensing that restricts DNA replication to once per cell cycle. During S phase MCM2-7 functions as part of the replicative helicase but within the pre-RC MCM2-7 is inactive. The organization of replicative DNA helicases before and after loading onto DNA has been studied in bacteria and viruses but not eukaryotes and is of major importance for understanding the MCM2-7 loading mechanism and replisome assembly. Lack of an efficient reconstituted pre-RC system has hindered the detailed mechanistic and structural analysis of MCM2-7 loading for a long time. We have reconstituted Saccharomyces cerevisiae pre-RC formation with purified proteins and showed efficient loading of MCM2-7 onto origin DNA in vitro. MCM2-7 loading was found to be dependent on the presence of all pre-RC proteins, origin DNA, and ATP hydrolysis. The quaternary structure of MCM2-7 changes during pre-RC formation: MCM2-7 before loading is a single hexamer in solution but is transformed into a double-hexamer during pre-RC formation. Using electron microscopy (EM), we observed that loaded MCM2-7 encircles DNA. The loaded MCM2-7 complex can slide on DNA, and sliding is not directional. Our results provide key insights into mechanisms of pre-RC formation and have important implications for understanding the role of the MCM2-7 in establishment of bidirectional replication forks.

ATPase-dependent cooperative binding of ORC and Cdc6 to origin DNA.

Binding of Cdc6 to the origin recognition complex (ORC) is a key step in the assembly of a pre-replication complex (pre-RC) at origins of DNA replication. ORC recognizes specific origin DNA sequences in an ATP-dependent manner. Here we demonstrate cooperative binding of Saccharomyces cerevisiae Cdc6 to ORC on DNA in an ATP-dependent manner, which induces a change in the pattern of origin binding that requires the Orc1 ATPase. The reaction is blocked by specific origin mutations that do not interfere with the interaction between ORC and DNA. Single-particle reconstruction of electron microscopic images shows that the ORC–Cdc6 complex forms a ring-shaped structure with dimensions similar to those of the ring-shaped MCM helicase. The ORC-Cdc6 structure is predicted to contain six AAA+ subunits, analogous to other ATP-dependent protein machines. We suggest that Cdc6 and origin DNA activate a molecular switch in ORC that contributes to pre-RC assembly.

Mechanism of origin unwinding: sequential binding of DnaA to double‐ and single‐stranded DNA

The initiator protein DnaA of Escherichia coli binds to a 9mer consensus sequence, the DnaA box (5′‐TTA/TTNCACA). If complexed with ATP it adopts a new binding specificity for a 6mer consensus sequence, the ATP‐DnaA box (5′‐AGatct). Using DNase footprinting and surface plasmon resonance we show that binding to ATP‐DnaA boxes in the AT‐rich region of oriC of E.coli requires binding to the 9mer DnaA box R1. Cooperative binding of ATP‐DnaA to the AT‐rich region results in its unwinding. ATP‐DnaA subsequently binds to the single‐stranded region, thereby stabilizing it. This demonstrates an additional binding specificity of DnaA protein to single‐stranded ATP‐DnaA boxes. Binding affinities, as judged by the DnaA concentrations required for site protection in footprinting, were ∼1 nM for DnaA box R1, 400 nM for double‐stranded ATP‐DnaA boxes and 40 nM for single‐stranded ATP‐DnaA boxes, respectively. We propose that sequential recognition of high‐ and low‐affinity sites, and binding to single‐stranded origin DNA may be general properties of initiator proteins in initiation complexes.

ATP– and ADP–DnaA protein, a molecular switch in gene regulation

DnaA protein functions by binding to asymmetric 9mer DNA sites, the DnaA boxes. ATP–DnaA and ADP–DnaA bind to 9mer DnaA boxes with equal affinity, but only ATP–DnaA protein binds in addition to an as yet unknown 6mer site, the ATP–DnaA box AGATCT, or a close match to it. ATP–DnaA protein binding to ATP–DnaA boxes is restricted to sites located in close proximity to DnaA boxes, suggesting that protein–protein interaction is required for its stabilization. We show that ATP–DnaA represses dnaA transcription much more efficiently than ADP–DnaA. DnaA is thus a regulatory molecule that, depending on the adenosine nucleotide bound, can bind to different sequences and thereby fulfill distinct functions.

Analysis of protein-DNA interactions using surface plasmon resonance

Protein-DNA interactions are required for access and protection of the genetic information within the cell. Historically these interactions have been studied using genetic, biochemical, and structural methods resulting in qualitative or semiquantitative interaction data. In the future the focus will be on high quality quantitative data to model a huge number of interactions forming a specific network in system biology approaches. Toward this aim, BIAcore introduced in 1990 the first commercial machine that uses surface plasmon resonance (SPR) to study the real-time kinetics of biomolecular interactions. Since then systems have been developed to allow for robust analysis of a multitude of protein-DNA interactions. Here we provide a detailed guide for protein-DNA interaction analysis using the BIAcore, starting with a description of the SPR technology, giving recommendations on preliminary studies, and finishing with extensive information on quantitative and qualitative data analysis. One focus is on cooperative protein-DNA interactions, where proteins interact with each other to modulate their binding specificity or affinity. The BIAcore has been used for the last 14 years to study protein-DNA interactions; our literature review focuses on some high quality studies describing a wide range of experimental uses, covering simple 1 : 1 interactions, analysis of complicated multiprotein-DNA interaction systems, and analytical uses.

Functional domains of DnaA proteins

Functional domains of the initiator protein DnaA of Escherichia coli have been defined. Domain 1, amino acids 1-86, is involved in oligomerization and in interaction with DnaB. Domain 2, aa 87-134, constitutes a flexible loop. Domain 3, aa 135-373, contains the binding site for ATP or ADP, the ATPase function, a second interaction site with DnaB, and is required for local DNA unwinding. Domain 4 is required and sufficient for specific binding to DNA. We show that there are three different types of cooperative interactions during the DNA binding of DnaA proteins from E. coli, Streptomyces lividans, and Thermus thermophilus: i) binding to distant binding sites; ii) binding to closely spaced binding sites; and iii) binding to non-canonical binding sites.

Bacterial replication initiator DnaA. Rules for DnaA binding and rolesof DnaA in origin unwinding and helicase loading

We review the processes leading to the structural modifications required for the initiation of replication in Escherichia coli, the conversion of the initial complex to the open complex, loading of helicase, and the assembly of two replication forks. Rules for the binding of DnaA to its binding sites are derived, and the properties of ATP-DnaA are described. We provide new data on cooperative interaction and dimerization of DnaA proteins of E. coli, Streptomyces and Thermus thermophilus, and on the stoichiometry of DnaA-oriC complexes of E. coli.

An ORC/Cdc6/MCM2-7 Complex Is Formed in a Multistep Reaction to Serve as a Platform for MCM Double-Hexamer Assembly

In Saccharomyces cerevisiae and higher eukaryotes, the loading of the replicative helicase MCM2-7 onto DNA requires the combined activities of ORC, Cdc6, and Cdt1. These proteins load MCM2-7 in an unknown way into a double hexamer around DNA. Here we show that MCM2-7 recruitment by ORC/Cdc6 is blocked by an autoinhibitory domain in the C terminus of Mcm6. Interestingly, Cdt1 can overcome this inhibitory activity, and consequently the Cdt1-MCM2-7 complex activates ORC/Cdc6 ATP-hydrolysis to promote helicase loading. While Cdc6 ATPase activity is known to facilitate Cdt1 release and MCM2-7 loading, we discovered that Orc1 ATP-hydrolysis is equally important in this process. Moreover, we found that Orc1/Cdc6 ATP-hydrolysis promotes the formation of the ORC/Cdc6/MCM2-7 (OCM) complex, which functions in MCM2-7 double-hexamer assembly. Importantly, CDK-dependent phosphorylation of ORC inhibits OCM establishment to ensure once per cell cycle replication. In summary, this work reveals multiple critical mechanisms that redefine our understanding of DNA licensing.

Cryo-EM structure of a helicase loading intermediate containing ORC-Cdc6-Cdt1-MCM2-7 bound to DNA.

In eukaryotes, the Cdt1-bound replicative helicase core MCM2-7 is loaded onto DNA by the ORC–Cdc6 ATPase to form a prereplicative complex (pre-RC) with an MCM2-7 double hexamer encircling DNA. Using purified components in the presence of ATP-γS, we have captured in vitro an intermediate in pre-RC assembly that contains a complex between the ORC–Cdc6 and Cdt1–MCM2-7 heteroheptamers called the OCCM. Cryo-EM studies of this 14-subunit complex reveal that the two separate heptameric complexes are engaged extensively, with the ORC–Cdc6 N-terminal AAA+ domains latching onto the C-terminal AAA+ motor domains of the MCM2-7 hexamer. The conformation of ORC–Cdc6 undergoes a concerted change into a right-handed spiral with helical symmetry that is identical to that of the DNA double helix. The resulting ORC–Cdc6 helicase loader shows a notable structural similarity to the replication factor C clamp loader, suggesting a conserved mechanism of action.

Cdc6 ATPase ACTIVITY REGULATES ORC-Cdc6 STABILITY AND THE SELECTION OF SPECIFIC DNA SEQUENCES AS ORIGINS OF DNA REPLICATION

DNA replication, as with all macromolecular synthesis steps, is controlled in part at the level of initiation. Although the origin recognition complex (ORC) binds to origins of DNA replication, it does not solely determine their location. To initiate DNA replication ORC requires Cdc6 to target initiation to specific DNA sequences in chromosomes and with Cdt1 loads the ring-shaped mini-chromosome maintenance (MCM) 2–7 DNA helicase component onto DNA. ORC and Cdc6 combine to form a ring-shaped complex that contains six AAA+ subunits. ORC and Cdc6 ATPase mutants are defective in MCM loading, and ORC ATPase mutants have reduced activity in ORC·Cdc6·DNA complex formation. Here we analyzed the role of the Cdc6 ATPase on ORC·Cdc6 complex stability in the presence or absence of specific DNA sequences. Cdc6 ATPase is activated by ORC, regulates ORC·Cdc6 complex stability, and is suppressed by origin DNA. Mutations in the conserved origin A element, and to a lesser extent mutations in the B1 and B2 elements, induce Cdc6 ATPase activity and prevent stable ORC·Cdc6 formation. By analyzing ORC·Cdc6 complex stability on various DNAs, we demonstrated that specific DNA sequences control the rate of Cdc6 ATPase, which in turn controls the rate of Cdc6 dissociation from the ORC·Cdc6·DNA complex. We propose a mechanism explaining how Cdc6 ATPase activity promotes origin DNA sequence specificity; on DNA that lacks origin activity, Cdc6 ATPase promotes dissociation of Cdc6, whereas origin DNA down-regulates Cdc6 ATPase resulting in a stable ORC·Cdc6·DNA complex, which can then promote MCM loading. This model has relevance for origin specificity in higher eukaryotes.